紅螢光碳點結合透明質酸於細胞標定之應用

應用於生物成像的有機螢光染劑具有激發光波長範圍狹窄、光源選擇性少、且發光強度衰減快的缺點。奈米碳點具有低毒性、光穩定性、抗光漂白及螢光可調等優點，近年來已被逐漸應用在光學造影上。本研究以微波加熱法將對苯二胺 (p-Phenylenediamine, pPD) 合成紅色螢光碳點 (red fluorescent carbon nanodots, RCDs)，並將透明質酸 (hyaluronic acid, HA) 接枝到 RCDs 上，形成帶有透明質酸之碳點 (hyaluronic acid red fluorescent carbon nanodots, HA-RCDs)。RCDs 的激發光波長為 474 nm，發散光位置則為 642 nm；HA-RCDs 以最佳激發波長 519 nm 激發，可得出發散光位置為 591 nm。以傅立葉轉換紅外線光譜儀測定 HA-RCDs，獲得醯胺鍵的特徵吸收峰。以原子力顯微鏡所測得的 RCDs 粒徑為 3.83 ± 1.25 nm，而 HA-RCDs 的粒徑增加為 5.47 ± 1.91 nm；而以穿透式電子顯微鏡所測得的 RCDs 的粒徑為 7.85 ± 0.88 nm，而 HA-RCDs 粒徑則為 19.30 ± 1.26 nm。以動態雷射光散射儀測定表面電位，得知 RCDs 帶正電，HA-RCDs 帶負電。從上述實驗結果證明 HA 確實有接上 RCDs。將間葉幹細胞 (Mesenchymal stem cells, MSCs) 與 RCDs 及 HA-RCDs 共培養，發現 RCDs 於 20 μg/mL 時，細胞數有降低的趨勢，而 HA-RCDs 於 1 mg/mL 對細胞仍不具有毒性。MSCs在攝入碳點後可發出紅色螢光；而添加 HA 會抑制 MSCs 對碳點的攝入。流式細胞儀分析顯示 HA-RCDs可被MSCs攝入的含量較高。活體螢光影像部分，RCDs 及 HA-RCDs 可累積在肝臟、腎臟及腋下淋巴結中，最後由尿液排出體外。綜合上述結果，本研究製備的 HA-RCDs 可提升其在水溶液中的穩定性，並可促進 MSCs 的攝入，且可透過腎臟代謝，是一個具有生物安全性的螢光追蹤劑。

關鍵字

紅螢光碳點；透明質酸；間葉幹細胞；細胞標定

並列摘要

Most of organic fluorescent dyes for biological imaging have the following shortcomings: narrow range of excitation wavelength, limited selection of the light source, and fast decay of fluorescence intensity. Carbon nanodots (CDs) have low toxicity, good photostability, resistance to photobleaching, and phototunable fluorescent property, which have used in optical imaging recently. Here, red fluorescent carbon nanodots (RCDs) were synthesized by microwave assisted pyrolysis of p-phenylenediamine (pPD). RCDs were further conjugated with hyaluronic acid (HA) by amide bond formation to produce hyaluronic acid red fluorescent carbon nanodots (HA-RCDs) as verified by Fourier-transform infrared spectroscopy analysis. RCDs emitted at the wavelength of 642 nm when excited at 474 nm. HA-RCDs emitted at the wavelength of 591 nm when excited at 519 nm. The size of RCDs was 3.83 ± 1.25 nm analyzed by Atomic Force Microscopy (AFM). The size of HA-RCDs was increased to 5.47 ± 1.91 nm by AFM. The size of RCDs was 7.85 ± 0.88 nm analyzed by Transmission Electron Microscope (TEM). The size of HA-RCDs was increased to 19.30 ± 1.26 nm by TEM. Dynamic light scattering (DLS) analysis revealed that RCDs had positive zeta potential whereas HA-RCDs had negative zeta potential. RCDs at the concentration higher than 20 µg/mL lowered cell viability of mesenchymal stem cells (MSCs). HA-RCDs at the concentration of 1 mg/mL had no cytotoxicity. MSCs exhibited red fluorescence after 24-hour incubation with RCDs and HA-RCDs. Pre-incubation with HA inhibited cellular uptake of CDs. Flow cytometry analysis found that the uptake of HA-RCDs was higher than that of RCDs. In vivo imaging system analysis (IVIS) showed that RCDs and HA-RCDs could accumulate in liver, kidney, and axillary lymph nodes and could be further metabolized through urinary system. In summary, HA-RCDs have good stability in aqueous solution, enhanced cellular uptake by MSCs, and can be excreted via kidney metabolism. This suggests that HA-RCDs are biocompatible fluorescence tracers.

並列關鍵字

red fluorescent carbon nanodot ； hyaluronic acid ； mesenchymal stem cell ； cell labeling

參考文獻

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紅螢光碳點結合透明質酸於細胞標定之應用

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